Novel methods of use of hsp70 for increased performance or treatment of hsp70 related disorders

ABSTRACT

The present invention relates to novel therapies that utilize exogenous HSP70 for the treatment of disorders or conditions regulated by HSP70 through administration of exogenous HSP70.

RELATED APPLICATIONS

This application is a US Utility Application that claims the benefit ofpriority from U.S. Provisional Patent Application No. 61/502,332, filedon Jun. 28, 2011, the entirety of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Over 6 million Americans are diagnosed annually with musculoskeletaldisease, which is a major cause of work disability. Annual costsassociated with treatment and care of musculoskeletal disease is in thetens of billions of dollars. Muscle degeneration and impairment in suchdisease may be stemming from various causes, including tissue ischemia,severe injury, advanced age, and muscular dystrophy resulting formgenetic defects as well as major diseases such as cancer or kidneyfailure.

Current key therapeutic approaches and standards of care are typicallylimited to physiotherapeutic rehabilitation, pain management andanti-inflammatory medications. Lately, there has been an increasedinterest in developing new interventions focused directly on muscleregeneration. For example, some promise was shown by using celltherapies and related muscle progenitor cells injected directly or as apart of the polymer scaffold (Levenberg et al. 2005). However, thisapproach is limited by poor integration of the transplanted cells andtheir low survival.

Growth factors have also been extensively explored in context ofischemic vascular regeneration (Silva et al., 2007), and more recentlyextended to muscular regeneration (Messina et al., 2007; Karkkainen etal., 2009). It is notable that most of the work was done with a vascularendothelial growth factor VEGF, a well-known proangiogenic regulator andthus it was challenging to separate angiogenic contributions from directeffects on muscle development and evolution of the related progenitorcells population. However, in a recent article Borselli et al. (Borselliet al., 2010) showed a synergistic effect of VEGF and an insulin growthfactor IGF-1 that binds to type I receptors and may activate severalintracellular signaling pathways including the MAP kinase, PI3-K, andthe calcium—calmodulin-dependent protein kinase pathway; and thereforelead to the activation and proliferation of the satellite cells, theirterminal myogenic differentiation, increased protein synthesis, andmyofiber survival. In this respect, it was shown that asustained-release alginate formulation of both singular agents and theircombination was much more effective than bolus administration.

Additional therapeutic approaches have looked into heat shock protein 70(HSP70), one of several proteins in the general class of heat shockproteins (HSPs). HSP70 has been implicated in many processes includingfolding and unfolding of nascent proteins, activation of amulti-enzymatic complex, and protein transport. Additionally, HSPs areimportant for the maintenance of cell integrity during normal growth aswell as during pathophysiological conditions (Vigh et al. 1997). It hasbeen shown that tissue injury, whether caused by surgery, trauma ordisease, results in the induction of heat shock/stress proteins. Aninducible form of the 70 kDa heat shock protein family HSP72 has beendetected intra- and extra-cellularly in different organs, includingskeletal muscles in response to exercise.

The biological significance of these processes appears to be related: toaid cell survival and chaperone misfolded and denatured proteins. Asmolecular chaperones, HSPs are also fundamental in facilitating cellularremodeling processes inherent to the training response (Morton et al.2009; Whitman et al. 2008). Moreover, the beneficial effects of HSPshave been implicated in a number of different diseases such as diabetes;wound healing (Atalay et al. Curr. Pep. Prot. Sc. 2009; 10:85); cancer(Ciocca et al., Stress Cell Chap. 2005; 10:86; Guzhova et al. Tsitologia2005, 47:187); sepsis (McConnell et al.; J. Immun. 2011; 186:3718;Kustanova et al. Cell Stress Chap. 2006; 11:276); cardiac injury(Knowlton et al. Am. J. Physiol. Heart Cir. Physiol. 2001; 280:H455);muscular injury and degeneration; recovery from physical and exercisestress (Morton et al. Sports Med. 2009; 39(8):643); neuro-degenerationincluding Parkinson disease, Alzheimer disease, Huntington disease,amyotrophic lateral sclerosis (Turturici et al., Biochem. Res. Int.2011); spinal cord injury (Reddy et al. Neurosurg. Focus 2008, 25(5):1);traumatic brain injury; stroke; eye neurodegenerative diseases includingglaucoma and macular degeneration (Levin, Surv. Ophthalm. 2003; 48:S21);and epilepsy (Ekimova et al. J. Neurochem. 2010; 115:1035).

At the same time, it has been found that patients with chronic fatiguesyndrome (CFS) present an accentuated exercise-induced oxidative stress.Compared with controls, resting CFS patients had low levels of HSP70 anddelayed and marked reduction of HSP70 levels in response to maximalexercise (Jammes et al. 2009). In this regard, HSP70 has been implied asa main mediator of the phytoadaptogens such as Rhodiola rosea andEleutheroccoccus senticosus that improve attention, cognitive functionand mental performance in fatigue and chronic fatigue syndrome as wellas increase endurance. HSP70 inhibits the expression of the NO synthaseII and affects the levels of circulating cortisol via direct interactionwith glucocorticoid receptors and JNK pathway. Consequently, preventionof the stress-induced NO and associated decrease in ATP productionresult in increased performance and endurance (Panossian et al. 2009).Additionally, in HSP70 inhibition (by siRNA interference and a smallmolecule, N-formyl-3,4-methylenedioxy-benzylidene-butyrolactam, KNK-437)models of vascular insufficiency (Shiota et al. 2010), the lack of HSP70was linked to disruption of the VEGF-related pathways and Akt activationspecifically.

In an effort to capitalize on the involvement of HSP70 in many of thesedisorders or conditions, several patent applications have reported theutility of HSPs in relation to the recovery from injury (Slepian, USPatent 5,914,345; Srivastava, US Patent Application US 2003/0012793).Additional applications have focused on compounds that induce HSP70,such as geranylgeranylacetone, which have been described to protectsubjects from the effect of ischemic-reperfusion injury (Takahashi N, USPatent Application 6,846,845 B2). Moreover, BGP-15, a pharmacologicalinducer of Hsp72 (currently in clinical trials for diabetes), has beenshown to improve muscle architecture, strength, and contractile functionin severely affected diaphragm muscles in two dystrophic mice modelsrelevant to Duchenne muscular dystrophy (Gehrig et al., 2012).

Although evidence has suggested the role of HSP70 in certainindications, current treatments that have adopted strategies to controlin vivo HSP70 production have not met the need in this arena. As such,there is strong need for novel therapies that address this currentdemand.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to novel therapies thatutilize HSP70 for the treatment of disorders or conditions regulated byHSP70 through administration of exogenous HSP70. In this regard, thepresent invention provides formulations of HSP70 that offer therapeuticeffectiveness in the treatment of HSP70 related disorders or conditions.Furthermore, the present invention is directed to methods comprising theadministration of exogenous HSP70 for use in the treatment of HSP70related disorders or conditions, for example, for increased performancecapability, e.g., physical and mental, increased endurance, oralleviating fatigue syndrome. In a particular example, the HSP70 relateddisorder or condition may be muscle degeneration and impairment stemmingfrom various causes including tissue ischemia, severe injury,exercise-induced tissue injury and fatigue, advanced age, musculardystrophy resulting form genetic defects and also caused by majordiseases such as cancer, kidney failure and others.

Thus, in one aspect, the invention provides a formulation comprisingHSP70, e.g., a therapeutically effective amount of HSP70, andpharmaceutically acceptable carrier.

In another aspect, the invention provides a method of treatment of anHSP70 related disorder or condition, wherein the method comprisesadministering to a subject a compound of the present invention, e.g., atherapeutically effective amount of HSP70, such that the HSP70 relateddisorder or condition is treated in the subject.

In another aspect, the invention provides a method to increaseperformance (e.g., endurance), alleviate a fatigue syndrome, or treatmuscle damage or muscle degeneration comprising administering to themammal a compound of the invention, e.g., a therapeutically effectiveamount of HSP70, e.g., human heat shock protein 70, such thatperformance (e.g., endurance) is increased, the fatigue syndrome isalleviated, or the muscle damage or muscle degeneration is treated inthe mammal.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts the variations in endurance (time of exercise toexhaustion) relative to the result of the first exercise session.

FIG. 2 depicts muscle electron microscopy (x15000) from the control(untreated) animal. Clearly visible is myofibrils separation and edema,capillary edema, and damaged erythrocytes (shadows) as manifestation ofthe rhabdomyolysis. An activated podocyte is located in the upper leftcorner.

FIG. 3 depicts muscle electron microscopy (x15000) from the treatedanimal (HSP70, 100 mcg). The view is dominated by an activated satellitecell with a large euchromatin-rich nucleus.

FIG. 4 depicts muscle electron microscopy (x15000) from the treatedanimal (HSP70-PEG, 100 mcg). The view is dominated by an activatedsatellite cell and numerous mitochondria.

FIG. 5 depicts muscle optical microscopy (x400) from the treated animal(HSP70, 100 mcg). Zones of the neoangiogenesis are clearly visible withpartial myofibrils separation.

FIG. 6 depicts muscle optical microscopy (x400) from the treated animal(HSP70-PEG, 100 mcg). Zones of the neoangiogenesis with largelymphocytes are clearly visible with partial myofibrils separation .

FIG. 7 depicts muscle electron microscopy (x15000) of the patient beforethe treatment with HSP70. Muscle damage is manifested in a formation ofthe vacuoles, delamination of the myofibrils and formation of theglycogen aggregates in between the myofibrils.

FIG. 8 depicts muscle electron microscopy (x15000) of the patient afterthe treatment with HSP70. Muscle regeneration is manifested in improvedcross-section, and activated myocyte nuclear function.

DETAILED DESCRIPTION OF THE INVENTION

The present provides a novel therapy for treatment of HSP70 relateddisorders or conditions. In particular, the present invention isdirected to HSP70, or formulations thereof, and the administration ofsuch for use in the treatment of HSP70 related disorders or conditions.The present invention, including compounds, methods, and pharmaceuticalcompositions will be described with reference to the followingdefinitions that, for convenience, are set forth below. Unless otherwisespecified, the below terms used herein are defined as follows:

I. Definitions

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

As used herein, the term “HSP70” or “heat shock protein 70,” which areused interchangeably herein, describes an exogenously derived heat shockprotein characterized by its association with the art-recognized familyof 70 kiloDalton heat shock proteins, the structures of which arewell-known in that art. In addition, as used herein, the term “HSP70”covers native HSP70, recombinant HSP70, as well as derivatives thereof,e.g., PEG-HSP70.

As used herein, “HSP70 related disorders or conditions” describes theclass of conditions or disorders that are regulated by HSP70. ExemplaryHSP70 related disorders or conditions include, but are not limited tomyopathy, both congenital such as muscle dystrophies, and acquired suchas rhabdomyolysis, polymyositis, and dermatomyositis; acute trauma;traumatic toxicosis due to crash injury; ischemia-reperfusion injuryincluding stroke and myocardial infarction; heart failure; muscle damageas a result of the excessive physical exercise, e.g., without anaboliceffects such as increase in body mass; cancer; neurodegenerativediseases such as Alzheimer's, Huntington's, Parkinson's, and Amyotropiclateral sclerosis; certain inflammation; reduced physical performance(e.g., endurance), e.g., relative to absolute capability of individual;reduced mental performance, e.g., relative to absolute capability ofindividual; fatigue syndrome; sleep deprivation; sepsis; and hemorrhagicshock. In a particular example, the HSP70 related disorder or conditionmay be muscle degeneration and impairment stemming from various causesincluding tissue ischemia, severe injury, exercise-induced tissue injuryand fatigue, advanced age, muscular dystrophy resulting form geneticdefects and also caused by major diseases such as cancer, kidney failureand others.

As used herein, the term “exogenous” describes the external origin ofthe HSP70, or alternatively stated, the exogenous HSP70 describedherein, is derived or developed from outside the subject, e.g., throughrecombinant methods.

As used herein, the term “endurance” describes the ability to sustain aphysical activity, perform repetitive submaximal contractions, or exerta force for a prolonged period. In certain embodiments, endurance mayinclude aerobic endurance, anaerobic endurance, speed endurance, andstrength endurance. In certain embodiments, increased endurancedescribes the ability to sustain the physical activity, contractions orforce for a increased periods of time or with less exerted effort.

As used herein, the term “performance” describes the manner or qualityof action taken, physically or mentally, to carry out a task. Increasedmental or physical performance refers to the improved ability to carryout that task or multiple sequential tasks. Moreover, the compounds ofthe present invention are intended to affect improvement in physicalperformance or mental performance. In one embodiment the performance isphysical performance. In certain embodiments where the performance isphysical, the improved physical performance may include increasedendurance, increased muscle power, or increased muscle strength. Inanother embodiment, the performance is mental performance. In certainembodiments where the performance is mental, the improved mentalperformance may include improved comprehension, improved memoryretention, and improved mental acuity.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” refer to the eradication or amelioration of adisease, disorder, or condition, or of one or more symptoms associatedwith the disease, disorder or condition. In certain embodiments, theterms refer to minimizing the advancement or worsening of the disease,disorder, or condition resulting from the administration of a compoundof the invention to a patient with such a disease, disorder, orcondition. In some embodiments, the terms refer to the administration ofa compound provided herein, with or without other additional activeagents, after the onset of symptoms of the particular disease, disorder,or condition. The terms “treating”, “treatment”, or the like, as usedherein covers the treatment of a disease, disorder, or condition in asubject, e.g., a mammal, and includes at least one of: (i) inhibitingthe disease, disorder, or condition, i.e., partially or completelyhalting its progression; (ii) relieving the disease, disorder, orcondition, i.e. causing regression of symptoms of the disease, disorder,or condition, or ameliorating a symptom of the disease, disorder, orcondition; and (iii) reversal or regression of the disease, disorder, orcondition, preferably eliminating or curing of the disease, disorder, orcondition. In a particular embodiment the terms “treating”, “treatment”,or the like, covers the treatment of a disease, disorder, or conditionin a mammal, e.g., a primate, e.g., a human, and includes at least oneof (i), (ii), and (iii) above. As is known in the art, adjustments forsystemic versus localized delivery, age, body weight, general health,sex, diet, time of administration, drug interaction and the severity ofthe condition may be necessary, and will be ascertainable with routineexperimentation by one of ordinary skill in the art.

As used herein, the terms “subject”, and “patient” are usedinterchangeably. The terms “subject” and “patient” refer to an animal(e.g., a bird such as a chicken, quail or turkey) or a mammal includingnon-primates (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat,cat, dog, and mouse) and primates (e.g., a monkey, chimpanzee and ahuman). In a particular embodiment, the subject is a human.

As used herein, and unless otherwise specified, the terms “prevent,”“preventing” and “prevention” refer to the prevention of the onset,recurrence or spread of a disease, disorder, or condition, or of one ormore symptoms thereof. In certain embodiments, the terms refer to theadministration of HSP70 to a subject, with or without other additionalactive compounds, prior to the onset of symptoms, particularly topatients at risk of a disease, disorder, or condition provided herein.The terms encompass the inhibition or reduction of a symptom of theparticular disease, disorder, or condition. Subjects with familialhistory of a disease, disorder, or condition, in particular, arecandidates for preventive regimens in certain embodiments. In addition,subjects who have a history of recurring symptoms are also potentialcandidates for the prevention. In this regard, the term “prevention” maybe interchangeably used with the term “prophylactic treatment.” Incertain embodiments, the prevention is achieved by administration of aprophylactically effective amount of a degradation resistant formulationof the invention.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of an active agent, e.g., HSP70, is an amountsufficient to provide a therapeutic benefit in the treatment ormanagement of a disease, disorder, or condition, or to delay or minimizeone or more symptoms associated with the disease, disorder, orcondition. A therapeutically effective amount of HSP70 means an amountof HSP70, alone or in combination with other therapies, which provides atherapeutic benefit in the treatment or management of the disease,disorder, or condition. The term “therapeutically effective amount” canencompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of disease, disorder, or condition, or enhances thetherapeutic efficacy of another therapeutic agent.

As used herein, and unless otherwise specified, the terms “manage,”“managing” and “management” refer to preventing or slowing theprogression, spread or worsening of a disease, disorder, or condition,or of one or more symptoms thereof. Often, the beneficial effects that asubject derives from a prophylactic and/or therapeutic agent do notresult in a cure of the disease, disorder, or condition. In this regard,the term “managing” encompasses treating a subject who had suffered fromthe particular disease, disorder, or condition in an attempt to preventor minimize the recurrence of the disease, disorder, or condition.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of an active agent, e.g., HSP70, is an amountsufficient to prevent a disease, disorder, or condition, or prevent itsrecurrence. A prophylactically effective amount of HSP70 means an amountof HSP70, alone or in combination with other agents, which provides aprophylactic benefit in the prevention of the disease. The term“prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

The term “polyethylene glycol” or “PEG” refers to a polyalkylene glycolcompound or derivative thereof, with or without coupling agents orderivatization with coupling or activating moieties. In its typicalform, PEG is a linear polymer with terminal hydroxyl groups and has theformula HO—CH₂CH₂ —(CH₂CH₂O)_(n)—CH₂CH₂—OH. The number of repeatingsubunits “n” in the PEG is approximated for the molecular mass describedin Daltons.

II. Compounds of the Invention

The compounds of the invention, HSP70 or derivatives thereof, may beused in the formulations and the methods of treatment of the invention.In certain embodiment, the invention provides formulations and methodsof use of HSP70 in its native form. In certain embodiment, the inventionprovides formulations and methods of use of HSP70 in a derivatized form,e.g., pegylated (PEG-HSP70). In certain embodiment, the inventionprovides formulations and methods of use of recombinant HSP70,recombinant human HSP70 (rhHSP70). In certain embodiment, the inventionprovides formulations and methods of use of derivatized recombinantHSP70, pegylated recombinant human HSP70 (PEG-rhHSP70).

In one embodiment, the present invention provides an isolated HSP70,e.g., suitable for administration to a subject. In certain embodiments,the HSP70 is in enriched form, i.e., enriched HSP70, wherein theenriched HSP70 comprises HSP70 substantially free of non-HSP70 material.In specific embodiments, such enriched HSP70 is greater than 50% pure(or less than 50% non-HSP70 material), e.g., is greater than 60% pure,e.g., is greater than 70% pure, e.g., is greater than 80% pure, e.g., isgreater than 90% pure, e.g., is greater than 92% pure, e.g., is greaterthan 94% pure, e.g., is greater than 96% pure, e.g., is greater than 98%pure, e.g., is greater than 99% pure.

III. Methods of the Invention

The invention relates to the method of use of a compound or formulationsof the invention, which contain a heat shock protein 70, e.g., arecombinant human heat shock protein 70, for the treatment of HSP70related disorders or conditions. For example, in one embodiment, theinvention provides a method of treatment of an HSP70 related disorder orcondition, wherein the method comprises administering to a subject,e.g., a mammal, a compound of the invention, e.g., a therapeuticallyeffective amount of HSP70 (e.g., human heat shock protein 70, e.g., aformulation of human heat shock protein 70), such that the HSP70 relateddisorder or condition is treated in the subject.

In particular embodiments of the invention, the HSP70 related disorderor condition is selected from the group consisting of myopathy, bothcongenital such as muscle dystrophies, and acquired such asrhabdomyolysis, polymyositis, and dermatomyositis; acute trauma;traumatic toxicosis due to crash injury; ischemia-reperfusion injuryincluding stroke and myocardial infarction; heart failure; muscle damageas a result of the excessive physical exercise, e.g., without anaboliceffects such as increase in body mass; cancer; neurodegenerativediseases such as Alzheimer's, Huntington's, Parkinson's, and Amyotropiclateral sclerosis; certain inflammation; reduced physical performance(e.g., endurance), e.g., relative to absolute capability of individual;reduced mental performance, e.g., relative to absolute capability ofindividual; fatigue syndrome; sleep deprivation; sepsis; and hemorrhagicshock. In a particular example, the HSP70 related disorder or conditionmay be muscle degeneration and impairment stemming from various causesincluding tissue ischemia, severe injury, exercise-induced tissue injuryand fatigue, advanced age, muscular dystrophy resulting form geneticdefects and also caused by major diseases such as cancer, kidney failureand others.

In a specific embodiment, the HSP70 related disorder is sarcopenia,e.g., as associated with aging and the progressive decline of musclemass, strength, and quality. In another specific embodiment, the HSP70related condition is reduced physical performance (e.g., endurance), orreduced mental performance, wherein the performance (or endurance) isincreased by treatment with the compounds of the invention. In anotherspecific embodiment, the HSP70 related disorder is fatigue syndrome. Inanother specific embodiment, the HSP70 related disorder or condition ismuscle damage or muscle degeneration. In another specific embodiment,the HSP70 related disorder or condition is cancer.

In another embodiment, the invention provides a method of treatment ofan HSP70 related disorder or condition, wherein the method comprisesadministering to a subject a therapeutically effective amount of HSP70.These conditions include myopathy, both congenital such as muscledystrophies, and acquired such as rhabdomyolysis, polymyositis, anddermatomyositis; acute trauma; traumatic toxicosis due to crash injury;ischemia-reperfusion injury including stroke and myocardial infarction;heart failure; muscle damage as a result of the excessive physicalexercise, e.g., without anabolic effects such as increase in body mass;cancer; neurodegenerative diseases such as Alzheimer's, Huntington's,Parkinson's, and Amyotropic lateral sclerosis; certain inflammation;reduced physical performance (e.g., endurance), e.g., relative toabsolute capability of individual; reduced mental performance, e.g.,relative to absolute capability of individual; fatigue syndrome; sleepdeprivation; sepsis; and hemorrhagic shock. In a specific embodiment,the HSP70 related disorder is sarcopenia, e.g., as associated with agingand the progressive decline of muscle mass, strength, and quality. Inanother specific embodiment, the HSP70 related condition is reducedphysical performance (e.g., endurance), or reduced mental performance,wherein the performance (or endurance) is increased by treatment with acompound of the invention. In another specific embodiment, the HSP70related disorder is fatigue syndrome. In another specific embodiment,the HSP70 related disorder or condition is muscle damage or muscledegeneration. In another specific embodiment, the HSP70 related disorderor condition is cancer.

In another embodiment, the invention provides a method to increaseperformance (e.g., endurance), alleviate fatigue syndrome, or treatmuscle damage or muscle degeneration comprising administering to asubject, e.g., a mammal, a compound of the invention, e.g., atherapeutically effective amount of HSP70 (e.g., human heat shockprotein 70, e.g., a formulation of human heat shock protein 70) suchthat performance is increased, the fatigue syndrome is alleviated, orthe muscle damage or muscle degeneration is treated in the subject.

In another embodiment, the invention provides a method to increaseperformance (e.g., endurance), alleviate a fatigue syndrome, or treatmuscle damage or muscle degeneration comprising administering to themammal a formulation comprising human heat shock protein 70, such thatperformance is increased, the fatigue syndrome is alleviated, or themuscle damage or muscle degeneration is treated in the mammal.

In another embodiment, the invention provides a method to treat cancercomprising administering to the mammal a compound of the invention,e.g., a formulation comprising human heat shock protein 70, such thatthe cancer is treated in the mammal.

In certain embodiments of the invention, the HSP70 is recombinant humanHSP70.

In certain embodiments of the invention, the HSP70 is pegylated HSP70.

In certain embodiments of the invention, the mammal is human.

In certain embodiments of the invention, the formulation is administeredintravenously, subcutaneously, intranasally, orally, or by inhalation.

In addition, the present invention is intended to include any novelmethod of preparation of the compounds or formulations of the presentinvention.

In an additional embodiment, the invention provides a method to increaseperformance capability, alleviate a fatigue syndrome, and treat muscledamage and muscle degeneration comprising administering to the mammal,e.g., a human, a composition comprising recombinant human heat shockprotein 70.

In another embodiment, the invention provides a method to increaseperformance capability, alleviate a fatigue syndrome, and treat muscledamage and muscle degeneration comprising administering to the mammal,e.g., a human, a composition comprising pegylated recombinant human heatshock protein 70. In certain embodiments, the recombinant human heatshock protein 70 shows a rate of clearance from serum that is at leastan order of magnitude slower than unpegylated protein.

Without wishing to be bound by theory, in certain embodiments it isbelieved that HSP70 of the present invention may confer its action bystimulating proangiogenesis and activating satellite cells - a keypopulation of the muscle stem cells.

IV. Formulations of the Invention

The formulations of the invention comprise HSP70, e.g., atherapeutically effective amount of HSP70, and may utilize anyadministration route that does not significantly impair the ability ofthe HSP70 to perform its intended function, e.g., intravenousinjections, oral administration, nasal administration, intravaginaladministration, rectal administration, or administration throughinhalation.

As such, one embodiment of the invention provides a formulationcomprising HSP70, e.g., a therapeutically effective amount of HSP70, andpharmaceutically acceptable carrier. In certain embodiments of theinvention, the formulation comprising a compound of the invention isformulated for intravenous, subcutaneous, intranasal, oral, orinhalation administration.

In certain embodiments of the invention, the amount of the heat shockprotein 70 present in the composition is in a range of 0.1 to 10milligrams.

In certain embodiments of the invention, the HSP70 is recombinant humanHSP70.

In certain embodiments of the invention, the HSP70 is pegylated HSP70,e.g., pegylated recombinant human HSP70, e.g., PEG-rhHSP70.

In certain embodiments, to prepare pharmaceutical formulationscontaining and HSP70 (e.g., PEG-rhHSP70) the HSP70 material, e.g., atherapeutically effective amount of the material, is admixed with apharmaceutically acceptable carrier or excipient. Preferably the carrieror excipient is inert. A pharmaceutical carrier can be any compatible,non-toxic substance suitable for delivering the HSP70 formulations ofthe invention to a subject. Examples of suitable carriers include normalsaline, Ringer's solution, dextrose solution, and Hank's solution.Non-aqueous carriers such as fixed oils and ethyl oleate may also beused. A preferred carrier is 5% dextrose/saline. The carrier may containminor amounts of additives such as substances that enhance isotonicityand chemical stability, e.g., buffers and preservatives. Formulations ofthe invention can be administered orally or injected into the body.Injections include but are not limited to intramuscular, subcutaneous,intradermal or intravenous. Alternatively, intra-articular injection orother routes could be used in appropriate circumstances. Whenadministered parenterally, PEGylated HSP70 is preferably formulated in aunit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutical carrier. See, e.g., Avis et al., eds.,Pharmaceutical Dosage Forms: Parenteral Medications , Dekker, N.Y.(1993); Lieberman et al., eds., Pharmaceutical Dosage Forms: Tablets ,Dekker, N.Y. (1990); and Lieberman et al., eds., Pharmaceutical DosageForms: Disperse Systems , Dekker, N.Y. (1990). Alternatively,compositions of the invention may be introduced into a patient's body byimplantable or injectable drug delivery system, e.g., Urquhart et al.Ann. Rev. Pharmacol. Toxicol . 24:199-236, (1984); Lewis, ed.,Controlled Release of Pesticides and Pharmaceuticals, Plenum Press, NewYork (1981); U.S. Pat. Nos. 3,773,919; 3,270,960; and the like. Thepegylated HSP70 can be administered in aqueous vehicles such as water,saline or buffered vehicles with or without various additives and/ordiluting agents. Preparation of such pharmaceutical compositions isknown in the art; see, e.g., Remington's Pharmaceutical Sciences andU.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton,Pa. (1984).

A. Administration of Formulations of the Present Invention

In general, however, the formulations of the invention that compriseHSP70 can be administered in a variety of dosage forms that do notaffect or significantly detract from the increased stability of theHSP70 (i.e., allowing such formulations to achieve their desiredfunction) including, but not limited to, a solid dosage form or in aliquid dosage form, an oral dosage form, a parenteral dosage form, anintranasal dosage form, a suppository, a lozenge, a troche, buccal, acontrolled release dosage form, a pulsed release dosage form, animmediate release dosage form, an intravenous solution, a suspension orcombinations thereof. The dosage can be an oral dosage form. The oraldosage form can be a tablet or a caplet. The formulations can beadministered, for example, by oral or parenteral routes, includingintravenous, intramuscular, intraperitoneal, subcutaneous, transdermal,airway (aerosol), rectal, vaginal and topical (including buccal andsublingual) administration. In one embodiment, the formulationscomprising the HSP70 are delivered to a desired site, such as the brain,by continuous injection via a shunt.

In another embodiment, the formulations of the invention can beadministered parenterally, such as intravenous (i.v.) administration.The formulations for administration will commonly comprise a solution ofHSP70 dissolved in a pharmaceutically acceptable carrier. Among theacceptable vehicles and solvents that can be employed are water andRinger's solution, an isotonic sodium chloride. In addition, sterilefixed oils can conventionally be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil can be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid can likewise be used in the preparation of injectables. Thesesolutions are sterile and generally free of undesirable matter. Theseformulations may be sterilized by conventional, well known sterilizationtechniques. The formulations may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents,e.g., sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of HSP70 inthese formulations can vary widely, and will be selected primarily basedon fluid volumes, viscosities, body weight, and the like, in accordancewith the particular mode of administration selected and the patient'sneeds. For i.v. administration, the formulation can be a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension can be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation can also be asterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In one embodiment, HSP70 can be administered by introduction into thecentral nervous system of the subject, e.g., into the cerebrospinalfluid of the subject. The formulations for administration will commonlycomprise a solution of HSP70 dissolved in a pharmaceutically acceptablecarrier. In certain aspects, HSP70 is introduced intrathecally, e.g.,into a cerebral ventricle, the lumbar area, or the cisterna magna. Inanother aspect, HSP70 is introduced intraocullarly, to thereby contactretinal ganglion cells.

The pharmaceutically acceptable formulations can be suspended in aqueousvehicles and introduced through conventional hypodermic needles or usinginfusion pumps. Prior to introduction, the formulations can besterilized with, preferably, gamma radiation or electron beamsterilization.

For oral administration, the compounds will generally be provided inunit dosage forms of a tablet, pill, dragee, lozenge or capsule; as apowder or granules; or as an aqueous solution, suspension, liquid, gels,syrup, slurry, etc. suitable for ingestion by the patient. Tablets fororal use may include the active ingredients mixed with pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents include, butare not limited to sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include, but are not limitedto starch and gelatin, while the lubricating agent, if present, willgenerally be magnesium stearate, stearic acid or talc. If desired, thetablets may be coated with a material such as glyceryl monostearate orglyceryl distearate, to delay absorption in the gastrointestinal tract.

Pharmaceutical preparations for oral use can be obtained throughcombination of HSP70 with a solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable additional compounds, if desired, to obtain tablets or drageecores. Suitable solid excipients in addition to those previouslymentioned are carbohydrate or protein fillers that include, but are notlimited to, sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredients is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active HSP70 doses.

For transmucosal administration (e.g., buccal, rectal, nasal, ocular,etc.), penetrants appropriate to the barrier to be permeated are used inthe formulation. Such penetrants are generally known in the art.

In particular, formulations of the invention may be administered to thenasal cavity in any suitable form. For example, the formulations of theinvention may be administered to the nasal cavity in the form of dropsor a spray, or the powders of the invention may be administered inaerosolized form. One method of administering the formulations of theinvention would be to use a spray device. Spray devices can be single(“unit”) dose or multiple dose systems, for example comprising a bottle,pump and actuator, and are available from various commercial sources,including Pfeiffer (Germany), Valois (France), Calmar (Germany),Ursatech (Germany), Bespak (UK) and Becton-Dickinson (USA).Electrostatic spray devices, such as described in U.S. Pat. No.5,655,517, are also suitable for the intranasal administration of theformulations of the invention.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate. Formulations suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate. For intramuscular,intraperitoneal, subcutaneous and intravenous use, the HSP70 willgenerally be provided in sterile aqueous solutions or suspensions,buffered to an appropriate pH and isotonicity. Suitable aqueous vehiclesinclude Ringer's solution and isotonic sodium chloride. Aqueoussuspensions may include suspending agents such as cellulose derivatives,sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wettingagent such as lecithin. Suitable preservatives for aqueous suspensionsinclude ethyl and n-propyl p-hydroxybenzoate.

The suppositories for rectal administration of HSP70 can be prepared bymixing the drug with a suitable non-irritating excipient, which is solidat ordinary temperatures but liquid at the rectal temperatures and willtherefore melt in the rectum to release the drug. Such materials arecocoa butter and polyethylene glycols.

The compounds can be delivered transdermally, by a topical route,formulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, or aerosols.

The HSP70 may also be presented as aqueous or liposome formulations.Aqueous suspensions can contain HSP70 in admixture with excipientssuitable for the manufacture of aqueous suspensions. Such excipientsinclude, but are not limited to a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil suspensions can be formulated by suspending HSP70 in a vegetableoil, such as arachis oil, olive oil, sesame oil or coconut oil, or in amineral oil such as liquid paraffin; or a mixture of these. The oilsuspensions can contain a thickening agent, such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents can be added to provide apalatable oral preparation, such as glycerol, sorbitol or sucrose. Theseformulations can be preserved by the addition of an antioxidant such asascorbic acid. As an example of an injectable oil vehicle, see Minto, J.Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical formulationscan also be in the form of oil-in-water emulsions. The oily phase can bea vegetable oil or a mineral oil, described above, or a mixture ofthese. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent.

In addition to the formulations described previously, the HSP70 may alsobe formulated as a depot preparation. Such long acting formulations maybe administered by implantation or transcutaneous delivery (e.g.,subcutaneously or intramuscularly), intramuscular injection or atransdermal patch. Thus, for example, the compounds may be formulatedwith suitable polymeric or hydrophobic materials (e.g., as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

For administration by inhalation, the compounds are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

In general a suitable dose will be in the range of 0.001 to 10 mg perkilogram body weight of the recipient per day, preferably in the rangeof 0.005 to 1 mg per kilogram body weight per day. The desired dose ispreferably presented once daily, but may be dosed as two, three, four,five, six or more sub-doses administered at appropriate intervalsthroughout the day.

The HSP70 can be administered as the sole active agent, or incombination with other known therapeutics to be beneficial in thetreatment of related HSP70 disorders or conditions. In any event, theadministering physician can provide a method of treatment that isprophylactic or therapeutic by adjusting the amount and timing of drugadministration on the basis of observations of one or more symptoms ofthe disorder or condition being treated.

Details on techniques for formulation and administration are welldescribed in the scientific and patent literature, see, e.g., the latestedition of Remington's Pharmaceutical Sciences, Maack Publishing Co,Easton Pa. (“Remington's”). After a pharmaceutical composition has beenformulated in an acceptable carrier, it can be placed in an appropriatecontainer and labeled for treatment of an indicated condition). Foradministration of the HSP70, such labeling would include, e.g.,instructions concerning the amount, frequency and method ofadministration.

EXEMPLIFICATION

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

I. Synthetic Preparations of HSP70 and -HSP-70-PEG Example 1Fermentation of rhHSP70

To prepare sufficient amounts of HSP70, 5 L of the E. coli expressionstrain HMS 174 (DE3) hHSP70 may be grown in animal free mediumcontaining 12 g phytone peptone, 60 g yeast extract, 40 g sodiumchloride, 1g methionine, and water to 5 liters. Before inoculation withexpression strain bacteria, the medium may be sterilized, andsupplemented with kanamycin to a final concentration of 30 mcg permilliliter. The medium may then be inoculated with one liter of anovernight culture of the HMS174(DE3) bacteria. The pH, level ofdissolved oxygen and temperature inside the fermenter reactor may becarefully monitored. When the culture reaches an optical density ofapproximately 1.2 (for example, approximately 2.5 hours afterinoculation), IPTG (isopropyl-B-D-thiogalactopyranoside) may be added toa final concentration of 1 mM to initiate the induction of expression ofrhHSP70. Growth may then be continued until an optical density ofapproximately 2.7 is attained (for example, after an additional 3.5hours following addition of IPTG). The fermented bacteria may then beharvested by centrifugation using standard techniques. The bacterialpellet may be re-suspended in 1 L of the 10 mmol PBS buffer (1 mmolEDTA, 10% glycerol). The re-suspended bacteria may then optionally befrozen on dry ice before further processing. To continue processing, thefrozen bacteria may be thawed if necessary, and then may be disrupted bypressure lysis. The resulting lysate may then be cleared and sterilefiltered.

Example 2 Purification of rhHSP70

A bacterial lysate comprising rhHSP70 may be subjected to the followingtwo-step purification protocol. The specific example provided utilizes500 mL of lysate; adjustments may be made for different lysate volumes.In the first step of the protocol, 500 mL of the cleared lysate,prepared as set forth above, may be diluted two-fold with 10 mM sodiumphosphate buffer pH 7.0. This 1 L of diluted and cleared bacteriallysate may be loaded onto a of DEAE Sephacel (Pharmacia) column (13 cmx15 cm) previously equilibrated with 2 column volumes of buffer A (10 mMsodium phosphate buffer pH 7.0, 20 mM sodium chloride, 10 mM ammoniumsulfate). The elution is carried out with two column volumes of buffer B(20 mM sodium phosphate buffer pH 7, 85 mM sodium chloride, 10 mMammonium sulfate). About 2 L of eluate is expected for recovery. Theeluate may then be diafiltered (buffer exchanged) against a 10-foldvolume of buffer C (20 mM sodium citrate pH 6.0, 100 mM sodium chloride,10 mM ammonium sulfate).

In the second step of the protocol, a 700 mL column (13 cmx5.5 cm) ATPagarose (Sigma-Aldrich) may be equilibrated in one column volume ofbuffer D (20 mM sodium citrate pH 6.0, 85 mM sodium chloride, 10 mMammonium sulfate). And the diafiltered solution may be run over the ATPagarose column. The column may then be washed with six column volumes ofbuffer D and finally eluted with 2 column volumes of buffer E (20 mMsodium citrate pH 6.0, 100 mM sodium chloride, 10 mM ammonium sulfate, 1mM magnesium acetate, 1 mM ATP). The eluted material, human rhHSP70having a purity of greater than 95 percent may be buffer exchanged intophosphate buffered saline and concentrated to a final concentration of10-20 mg/ml, and then sterile filtered.

Example 3 PEGylation Techniques

The efficacy of a therapeutic agent may be enhanced by improving itsbioavailability via several means, one of which is PEGylation, a processof chemically linking polyethylene glycol (PEG) to the therapeutic agentof interest, with the resulting conjugate exhibiting an increased serumhalf-life. Additional advantages of the PEGylated products may alsoinclude lower immunogenicity, decreased dosing frequency, increasedsolubility, enhanced stability, and reduced renal clearance. Because themost common reactive sites on proteins (including peptides) forattaching PEG are the c amino groups of lysine and the a amino group ofthe N-terminal residue, early methods of PEGylation resulted inmodification of multiple sites, yielding not only monoPEGylatedconjugates consisting of mixtures of positional isomers, such asPEGINTRONTM (Grace et al., J. Biol. Chem. 2005; 280:6327) and PEGASYS®(Dhalluin et al., Bioconjugate Chem. 2005; 16:504), but also adductscomprising more than one PEG chain.

The PEG reagents that are used to prepare PEGylated compounds of theinvention may comprise a heterogenous mixture of PEGs having a differentnumber (n) of ethylene glycol subunits in the PEG polymer. A singleethylene glycol subunit (—(CH₂CH₂O)) of PEG has a molecular weight ofabout 44 Daltons. Therefore, the molecular weight of the PEG polymerdepends on the number (n). The PEGs attached to the PEGylated rhHSP70compounds of the present invention will have n in the range from about400 to about 1000 subunits. Preferably, the PEGs attached to thePEGylated HSP70 compounds of the present invention will have n in therange from about 400 to about 750.

A. Activated PEG for Amino or Imino Conjugation

To conjugate PEG to HSP70, an activated linker covalently attached toone or more PEG molecules may be reacted with an amino or imino group ofan amino acid residue, e.g., with an alpha amino group at the N-terminusof HSP70, to form a PEG-HSP70 of the present invention.

A linker is “activated” if it is chemically reactive and ready forcovalent attachment, e.g., to an amino group on an amino acid residue.Any activated linker can be used in this invention provided it canaccommodate one or more PEG molecules and form a covalent bond, e.g.,with an amino group of an amino acid residue under suitable reactionconditions. For example, the activated linker attaches to an alpha aminogroup in a highly selective manner over other attachment sites, e.g.,epsilon amino group of lysine or imino group of histidine.

Activated PEG can be represented by the formula: (PEG)_(b)-L′, where PEGcovalently attaches to a carbon atom of the linker to form an etherbond, b is 1 to 9 (i.e. 1 to 9 PEG molecules can be attached to thelinker), and L′ contains a reactive group (an activated moiety) that canreact with an amino or imino group on an amino acid residue to provide acovalent attachment of the PEG to HSP70.

In one embodiment, an activated linker (L′) of the invention contains analdehyde of the formula RCHO, where R is a linear (straight chain) orbranched C₁₋₁₁ alkyl. After covalent attachment of an activated linkerto HSP70, the linker (referred to as “-L-” in the structural formulasrecited herein) between the HSP70 and PEG contains 2 to 12 carbon atoms.Propionaldehyde is an example of an activated linker of this invention.PEG-propionaldehyde, and is described in U.S. Pat. No. 5,252,714 and iscommercially available from Shearwater Polymers (Huntsville, Ala);represented by the formula PEG-CH₂CH₂CHO.

In certain embodiments, a suitable activated branched (also known as“multi-armed”) linker can be used. Any suitable branched PEG linker thatcovalently attaches two or more PEG molecules to an amino group on anamino acid residue of HSP70, e.g., to an alpha amino group at theN-terminus, can be used. A branched linker used in this invention maycontain two or three PEG molecules. For example, a branched PEG linkerused in this invention can be a linear or branched aliphatic group thatis hydrolytically stable and contains an activated moiety, e.g., analdehyde group, which reacts with an amino group of an amino acidresidue, as described above. The aliphatic group of a branched linkermay contain 2 to 12 carbons. For example, an aliphatic group can be at-butyl which contains as many as three PEG molecules on each of threecarbon atoms (i.e., a total of 9 PEG molecules) and a reactive aldehydemoiety on the fourth carbon of the t-butyl. Examples of activated,branched PEG linkers are also described in U.S. Pat. Nos. 5,643,575,5,919,455, and 5,932,462. One having ordinary skill in the art, in lightof the present invention, can prepare modifications to branched PEGlinkers as desired, e.g., addition of a reactive aldehyde moiety.

Methods for the preparation of linkers for use in the present inventionare well known in the art, e.g., see U.S. Pat. Nos. 5,643,575,5,919,455, and 5,932,462. Activated PEG-linkers, such as PEG-aldehydes,can be obtained from a commercial source, e.g., Shearwater Polymers,(Huntsville, Ala.) or Enzon, Inc. (Piscataway, N.J.).

B. Conjugation Reaction Between PEG and HSP70 Amino or Imino Group

The following schematic illustrates a reaction between an activated PEGaldehyde linker and an amino or imino group of an amino acid residue ofone of the HSP70 monomers: PEG-R—CHO+NH₂—HSP70⇄PEG-R—CH=N—HSP70, where Ris a C₁₋₁₁ alkyl and N is nitrogen of a reactive amino group on an aminoacid residue of HSP70. In this reaction, the activated PEG covalentlyattaches to the HSP70 to form an imine linkage. Reduction of the iminelinkage by the reducing agent, e.g., sodium cyanoborohydride(Sigma-Aldrich, St. Louis, Mo.) forms pegylated HSP70, as shown:PEG-R—CH═N—HSP70+NaCNBH₃→PEG-R—CH₂—NH—HSP70. Other reducing agents canbe used instead of sodium cyanoborohydride in this reaction, includingsodium borohydride, tertiary butyl amine borane, sodium triacetylborohydide, dimethylamine borate, trimethylamine borate, and pyridineborate. Sodium cyanoborohydride is used in certain embodiments becauseit specifically reduces an imine linkage, which is formed between analdehyde group of the activated PEG and amino group of the amino acid ofHSP70.

As shown in the reactions, a Schiff base is formed during thepreparation of PEG-HSP70. The concern that this intermediate, which isvery difficult to separate from PEG-HSP70 and could lower the purity ofthe PEG-HSP70 if the intermediate accumulates in the reaction and is notreduced to the product, may be avoided by using higher concentrations ofreducing agent, about 75:1 to 350:1 (e.g., Kinstler et al., Pharm. Res.13:996-1002 (1996) and Chamow et al., Bioconjugate Chem. 5: 133-140(1994)).

Various aqueous buffers can be used in the present method to catalyzethe covalent addition of PEG to HSP70. The pH of a buffer used is fromabout 5.5 to 7.8, e.g., the pH is in a neutral range, e.g., from about6.3 to 7.5. This neutral pH range would also increase the site-specificpegylation of HSP70 at the alpha amino group of the N-terminus versusother imino or amino groups of other amino acid residues, e.g., lysineor histidine. In certain embodiments, a buffers having a pKa close toneutral pH range is used, e.g., phosphate buffer.

The temperature range for preparing a PEG-HSP70 of the invention is fromabout 5° C. to 30° C. For example, in certain embodiments, thetemperature is from about 18° C. to 25° C.

The pegylation reaction can proceed from 3 to 48 hours, e.g., 10 to 24hours. The reaction can be monitored using SE (size-exclusion)-HPLC,which can distinguish HSP70, mono-PEG-HSP70 and di-PEG-HSP70 (i.e.,pegylation occurs on two amino acid residues of HSP70), etc. At anytimewhen the desired HSP70 is obtained, the reaction can be terminated byadding glycine solution to quench any remaining activated PEG.

Conventional separation and purification techniques known in the art canbe used to purify PEG-HSP70, such as size exclusion (e.g. gelfiltration) and ion exchange chromatography.

It may be desirable to polish or resolve a population of PEG-HSP70 in aPEG-HSP70 composition prepared according to a method of the presentinvention. The polishing step separates less stable PEG-HSP70 (e.g.His-PEG-HSP70) from stable PEG-HSP70 (e.g. N-terminus-PEG-HSp70 orLys-PEG-HSP70), and thus can achieve greater homogeneity of stablepositional isomers, e.g., greater than 95% of a PEG-HSP70 composition.Less stable positional isomers of PEG-HSP70, e.g., histidine-PEG-HSP70,can be hydrolyzed during a polishing step. The population of PEG-HSP70can be incubated in an aqueous buffer, e.g., a TRIS buffer (e.g., 10 to300 mM, e.g., about 30 to 70 mM), at about pH 5.0 to 9.0, e.g., pH 7.0to 8.0 at 15° C. at 35° C. overnight. Alternatively, the population ofPEG-HSP70 can be treated with 0.05 to 0.4 M hydroxylamine HC1 salt (pHabout 6.5) at room temperature for 0.5 to 10 hours. Hydrolyzed HSP70 andPEG remnant can be removed from the population of stable PEG-HSP70 by aseparation/purification step using, e.g., gel filtration or ion exchangechromatography.

In U.S. Pat. No. 5,985,265 the PEGylation of interferon using analdehyde linker was accomplished at acidic pH 4.0 at 4° C. Conventionalwisdom in the art teaches that for most activated PEG, as the reactionpH is increased under basic conditions pegylation occurs at more stablesites on the protein. For PEGylated IL-10 (U.S. Pat. No. 7,052,686),succinimidyl carbonate-PEG forms about 90% Lys-PEG-IL-10 (more stable)and about 10% His-PEG-IL-10 (less stable) at a reaction pH 8.8, andabout 64% Lys-PEG-IL-10 and 36% His-PEG-IL-10 at a reaction pH 6.3.

C. Preparation of Cysteine Modified HSP70-PEG

In one embodiment, rhHSP70-PEG was prepared by using PEG-Maleimide-20KDa reagent (Nanocs, N.Y., USA). The maleimide group reactedspecifically with sulfhydryl groups when the pH of the reaction mixturewas between pH 6.5 and 7.5; the result was formation of a stablethioether linkage that was not reversible (i.e., the bond cannot becleaved with reducing agents). HSP70 has 5 cysteine groups in thefollowing AA positions: 17, 267, 306, 574, and 603.

No cysteine thiols were known to participate in the formation of theintramolecular disulfide bonds and therefore all cysteines arepotentially amenable to pegylation though in certain condition as Cys574had been reported having a propensity for forming intermoleculardisulfide bonds (Nemoto et al. 2006). From the structural consideration,Cys267, Cys306 could be buried in the middle of the protein and/or bemasked by substrate making them less likely to be accessible. The Ellmantest confirmed the presence of free thiol groups. However, whenconducting the pegylation reaction in PBS buffer at pH 7.1, no changesin HPLC retention time were seen and subsequent SDS-PAGE gels did notreveal any reaction products but only a spot at 70 kDA for the unalteredHSP70.

After multiple trials, PEGylation only proceeded using denatured proteinand the optimum conditions were in 6 M guanidine, whereas lowerguanidine concentrations or using urea led to less efficient coupling.By varying the ratio of PEG-Maleimide the extent of cysteine pegylationwas controlled, from an average of 1 to 5 PEG fragments per one proteinmolecule. Based on the HPLC data, a peak related to the starting proteinvanishes when using a total of 50 eqv of PEG-maleimide. The finalpurification of HSP70-PEG is achieved by size exclusion chromatographyin PBS at pH 7.4.

Pegylated protein products refolded rapidly when diluted in PBS orduring a passage through the size-exclusion column. For in vivoexperiment pegylated products were concentrated using an Amicon membrane(cutoff 4 kDa) to a concentration of 0.5-2 mg/ml (as determined by microBCA) and passed through a 0.22 micron filter. The biological activity ofthe HSP70-PEG molecules was confirmed by the ATP-ase test using acommercial kit (ENZO Life Sciences, Farmingdale, N.Y.). Differentco-factors, e.g., HSP40 and modified albumins, have been reported toimprove the test response; however, the most robust results werereceived in 1 mmol ADP.

II. In Vivo Applications Example 4 Application of the rhHSP70 forIncreased Performance Capability Experienced During Intense PhysicalExercise

The goal of this experiment was to study the role of exogenous rhHSP70in experimental model of intense physical exercise in healthy adult wildwhite male rats (180-220 g, “Rappolovo” animal facility) and its effecton increased endurance. rhHSP70 was used in two doses of 50 and 100 mcgper animal injected intraperitoneally in lml of water. The experimentwas conducted in 3 groups of animals: 6 animals (control, placeboinjection), 6 animals (50 mcg HSP70), 6 animals (100 mcg HSP70).Experimental animals were pre-selected from the group that passed aninitial fitness and training test: a daily run on a treadmill for 5 minrepeated for 3 consecutive days (treadmill rate 20 m/min, attack angle20°). The study treadmill test was run to exhaustion (termination of therunning routine by an animal) for a total of 9 sessions that wererepeated every second day, at the same time of the day. HSP70 wasadministered 5-6 min following the completion of the test for the first6 sessions. The observed animal running time was used as a measure ofendurance capability (FIG. 1, Table 1).

TABLE 1 Variations in performance (time of exercise to exhaustion)relative to the result of the first exercise session. Animal GroupsExercise Days of HSP70, Session No. Observation Control HSP70, 50 mcg100 mcg 1 1 100 100 100 2 3 105.6 ± 5.9  108.6 ± 4.8  113.0 ± 4.0  3 5106.2 ± 3.0  115.1 ± 5.9  119.0 ± 4.8* 4 8 109.9 ± 4.2  124.1 ± 5.1* 127.3 ± 5.2* 5 10 81.2 ± 3.7 111.7 ± 3.5*  113.9 ± 3.4* 6 12 61.9 ± 4.4104.2 ± 5.9*  121.5 ± 5.3* 7 15 55.6 ± 4.9 97.3 ± 3.0* 114.6 ± 4.6* 8 1747.7 ± 4.0 62.2 ± 3.3*  87.0 ± 4.0* 9 19 46.7 ± 3.1 61.1 ± 4.1*  82.2 ±3.8* Note: *statistically significant (p ≦ 0.05) compared to control

A statistically significant differentiation in the endurance capabilitywas seen during the 3rd training session: the control group and low-doseHSP70 animals were shown to have results similar to the training sessionNo.1, whereas the high-dose HSP70 animals demonstrated improvedendurance (Fig.1). In all the subsequent training sessions, theHSP70-treated animal outperformed a control group in a dose-dependentmanner.

Starting from the training session No. 5, the performance/endurance inall groups began to decrease. However, the trend was slower in theHSP70-treated animals. After termination of the HSP70 administrationfollowing the training session No. 6, the endurance capability hasfurther decreased, however, in each training session the animalreceiving HSP70 outperformed untreated animals, by as much as 35.5% inthe 100 mcg group in the last training session No.9.

As a result of the exhaustion following the exercise on the day 19, thecontrol group demonstrated increased levels of blood proteins,creatinine, as well as sodium and potassium as compared with bothtreated groups (Table 2). Additionally, treated animals displayeddecreased activity of the creatinine kinase and lactate dehydrogenase(LDG) that may indicate cytoprotective properties of the HSP70.Importantly, we detected no anabolic effects of HSP70—there was noadditional body mass increase in treated group as compared to thecontrol group though all animals demonstrated slightly elevated bodymass at the end of the experiment.

TABLE 2 Blood biochemistry results following completion of the study(day 19). Animal Groups Test Units Control HSP70, 50 mcg HSP70, 100 mcgProteins g/L 72.5 ± 1.7  63.6 ± 1.1* 66.0 ± 1.6* Creatinine mmol/L 67.7± 3.1  52.0 ± 3.5* 56.3 ± 1.4* Uric Acid mmol/L 9.1 ± 1.2 8.6 ± 0.7 9.7± 0.4 Glucose mmol/L 7.2 ± 0.5 6.6 ± 0.2 6.6 ± 0.8 K⁺ mmol/L 6.6 ± 0.36.8 ± 0.2 7.3 ± 0.2 Na⁺ mmol/L 149.4 ± 0.9  144.2 ± 0.5*  145.2 ± 0.5* Ca²⁺ mmol/L 2.50 ± 0.03 2.46 ± 0.05 2.46 ± 0.01 Cl⁻ mmol/L 112.2 ± 0.9 107.8 ± 1.2*  109.8 ± 0.3*  Creatinine kinase IU/L 13368.0 ± 455.3 10668.4 ± 312.0*  8954.5 ± 826.4* LDG IU/L 6245.7 ± 260.4  3146.8 ±227.2* 2407.8 ± 241.2* Note: *statistically significant (p ≦ 0.05)compared to control

Microscopy Analysis

To confirm the findings from the physiological functional testing andblood biochemistry, a limb muscle biopsy was conducted in the sacrificedanimals following a completion of the study. Transmission electronmicroscopy was carried out on a JEOL 110 CX and Hitachi H-300 electronmicroscopes using samples fixed by 2.5% glutaric aldehyde withsubsequent treatment in PBS solution of the osmium tetraoxide anddewatering in alcohol/acetone (FIG. 2-4). Tissue slices were generatedusing Ultracut-Reichert microtome. The Mallory staining for opticalmicroscopy was done with eosin and hematoxylin (FIG. 5,6).

In all the groups structural alterations were detected indicative of amuscle dystrophy, rhabdomyolysis and inflammation that was lesspronounced in the treated groups. An activation of angiogenesis is knownto be strongly associated with muscle regeneration (Borselli et al.,2010), with a capillary network being an important indication of therecovery processes. Correspondingly, in HSP70-treated animals, asignificant dose-dependent increase was found in the density of thecapillary vessels, by 170-330%, from 275/mm² in the control group to907/mm² in the high-dose HSP70 treated group (Table 3).

Additionally, staining was performed for the satellite cells, an adultstem cell population associated with myofibers and localized within thebasal lamina of the muscle fibers that are believed to be primarilyresponsible for muscle regeneration (Wagers et al., 2005). A 6-8 timesincrease was observed in the density of the satellite cells in theHSP70-treated versus untreated animals (Table 3).

TABLE 3 Capillary density and satellite cells count following completionof the exercise study. Animal Groups Parameter Control HSP70 (100 mcg)HSP70-PEG (100 mcg) Capillary 275.0 ± 21.2 907.5 ± 37.4*   990 ± 37.4*density (per mm²) Satellite cells 100  680.2 ± 150.3*  840.1 ± 170.4* (%control) Note: *statistically significant (p ≦ 0.05) compared to control

Example 5 Application of the rhHSP70-PEG for Increased PerformanceCapability Experienced During Intense Physical Exercise

The goal of this experiment was to study the role of exogenousrhHSP70-PEG in experimental model of intense physical exercise inhealthy adult wild white male rats (180-220 g, “Rappolovo” animalfacility) and its effect on increased endurance. rhHSP70-PEG was used ina dose of 100 mcg per animal injected intraperitoneally in lml of water.The experiment was conducted in 3 groups of animals: 6 animals (control,placebo injection), 6 animals (100 mcg HSP70), 6 animals (100 mcgHSP70-PEG). Experimental animals were pre-selected from the group thatpassed an initial fitness and training test: a daily run on a treadmillfor 5 min repeated for 3 consecutive days (treadmill rate 20 m/min,attack angle 20°). The study treadmill test was run to exhaustion(termination of the running routine by an animal) for a total of 10sessions that were repeated every day, at the same time of the day.HSP70 and HSP70-PEG were administered 5-6 min following the completionof the test on the daily basis. The observed animal running time wasused as a measure of performance capability (Table 4).

There was no significant difference between the groups in the first fivetraining sessions. After that, a ca. 30% drop was observed in theexercise time for the control and subsequent decrease in the follow-upsessions, to ca. 50% of the capacity at the end of the experiments. Incontrast, treated groups displayed a gradually increased performancereaching 115-125% as compared to the beginning of the study. HSP70-PEGshowed a statistically significant performance improvement over HSP70.This observation was corroborated by the higher density of the capillaryvessels and satellite cells count (Table 3).

Notably, we again detected no anabolic effects of HSP70 or HSP70-PEG(Table 5)—there was no additional body mass increase in treated group ascompared to the control group though all animals demonstrated slightlyelevated body mass at the end of the experiment (Table 5).

TABLE 4 Variations in performance (time of exercise to exhaustion)relative to the result of the first exercise session for HSP70-PEG.Exercise Animal Groups session/day Control HSP70 (100 mcg) HSP70-PEG(100 mcg) 1 100 100 100 2 105.3 ± 6.0 104.9 ± 6.8 103.4 ± 1.2 3 109.9 ±5.4 103.1 ± 6.6 104.6 ± 7.7 4 106.8 ± 8.5 119.1 ± 2.9 109.9 ± 5.5 5101.7 ± 6.6 112.9 ± 4.8 115.0 ± 1.8 6  86.9 ± 9.1 115.9 ± 5.2* 117.0 ±2.1* 7  56.1 ± 7.9 123.8 ± 3.8* 121.2 ± 5.6* 8  68.7 ± 4.1 119.2 ± 2.8*123.4 ± 3.6* 9  60.2 ± 1.7 117.0 ± 4.6* 125.1 ± 1.9* 10  54.8 ± 5.3*115.6 ± 36* 124.3 ± 6.7* Note: *statistically significant (p ≦ 0.05)compared to control

TABLE 5 Body mass change (g) in the course of exercise routine. AnimalGroups HSP70-PEG Exercise Session Control HSP70-PEG (100 mcg) (100 mcg)1 161.7 ± 6.9 162.2 ± 7.7 161.0 ± 7.8 2 166.3 ± 7.7 184.3 ± 7.1 179.3 ±3.5 3 168.0 ± 7.8 187.0 ± 7.1 183.0 ± 3.9 4 171.8 ± 8.2 190.0 ± 6.8187.3 ± 4.5 5 174.3 ± 8.2 189.8 ± 7.4 189.3 ± 4.1 6 174.2 ± 8.7 189.8 ±6.0 188.0 ± 2.9 7 174.8 ± 8.9 192.5 ± 5.6 189.3 ± 3.6 8 178.5 ± 8.7195.2 ± 4.8 184.0 ± 4.3 9 182.5 ± 8.8 198.2 ± 4.6 195.0 ± 3.7 10 182.2 ±8.6 196.0 ± 4.2 199.7 ± 4.1

Example 6 Clinical Use of HSP70 For Treatment of the Acquired MuscleMyopathy

Subject X, a 62 year old human patient suffered from a significant bodymass loss (35 kg over a period of 1 year), muscle atrophy of the lowerextremities and upper body, decrease in the muscle power and endurance,muscle contractures. Electron microscopy (Fig.7) of the biopsy frommusculus brachialis and gastrocnemius revealed significantrhabdomiolysis, mitochondrial damage with a formation of numerousvacuoles, and reduction in the number of satellite cells. The clinicaldiagnosis was established in Saint Petersburg (Russia) Military MedicalAcademy as an acquired mitochondrial myopathy and resulting cachexia.

HSP70 was then administered IV, 0.5 mg per dose over a period of 7 days.

Analysis following the 6 weeks subsequent to the last administration ofHSP70 showed that the patient's body mass had increased by 6.5 kg, whichwas associated with increased muscle strength. In addition, the electronmicroscopy (FIG. 8) revealed significant reduction in the myofibrildelamination and their improved cross-section, restoration of themitochondrial structure and activation of the myocyte nuclear function.

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INCORPORATION By REFERENCE

The entire contents of all patents, published patent applications andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents were consideredto be within the scope of this invention and are covered by thefollowing claims. Moreover, any numerical or alphabetical rangesprovided herein are intended to include both the upper and lower valueof those ranges. In addition, any listing or grouping is intended, atleast in one embodiment, to represent a shorthand or convenient mannerof listing independent embodiments; as such, each member of the listshould be considered a separate embodiment.

What is claimed is:
 1. A formulation comprising HSP70, andpharmaceutically acceptable carrier.
 2. The formulation of claim 1comprising a therapeutically effective amount of HSP70.
 3. Theformulation of 1, formulated for intravenous, subcutaneous, intranasal,oral, or inhalation administration.
 4. The formulation of any one ofclaims 1, wherein the HSP70 is recombinant human HSP70.
 5. Theformulation of any one of claims 1, wherein the HSP70 is pegylatedHSP70.
 6. The formulation of any one of claims 1, wherein the amount ofthe heat shock protein 70 present in the composition is in a range of0.1 to 10 milligrams.
 7. A method of treatment of an HSP70 relateddisorder or condition, wherein the method comprises administering to asubject a therapeutically effective amount of HSP70, such that the HSP70related disorder or condition is treated in the subject.
 8. A method toincrease performance, alleviate fatigue syndrome, or treat muscle damageor muscle degeneration comprising administering to a mammal atherapeutically effective amount of HSP70, such that performance isincreased, the fatigue syndrome is alleviated, or the muscle damage ormuscle degeneration is treated in the mammal.
 9. The method of claim 7,wherein the HSP70 human heat shock protein
 70. 10. The method of claim7, wherein the HSP70 is pegylated HSP70.
 11. The method of claim 7,wherein the mammal is human.
 12. The method claim 7, wherein theformulation is administered intravenously, subcutaneously, intranasally,orally, or by inhalation.
 13. The method of claim 8, wherein the HSP70human heat shock protein
 70. 14. The method of claim 8, wherein theHSP70 is pegylated HSP70.
 15. The method of claim 8, wherein the mammalis human.
 16. The method claim 8, wherein the formulation isadministered intravenously, subcutaneously, intranasally, orally, or byinhalation.